Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data?

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera fro...

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“…We chose to use relative read count data as a proxy for arthropod abundance, as supported by Piñol, Senar, and Symondson () and Deagle et al. ().…”

Section: Methodsmentioning

confidence: 99%

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

et al. 2018

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“…There is also the possibility of these methods over‐estimating prey taxa through secondary consumption, which, along with cannibalism, is nearly impossible to detect (Nielsen et al ., ), yet can be a significant potential source of error (Sheppard et al ., ). Furthermore, dietary metabarcoding data often contain biases such that there exists high uncertainty around quantitative estimates and in many instances biomass of prey cannot be inferred (Deagle et al ., ; Lamb et al ., ), whereas the relative fullness and frequency of occurrence methods are favoured and recommended for the standardization of feeding studies for their ability to discern relative prey diversity and abundance (Amundsen & Sánchez‐Hernández, ).…”

Section: Discussionmentioning

confidence: 99%

Diet composition of starry smooth‐hound Mustelus asterias and methodological considerations for assessing the trophic level of predatory fish

Phillips

Grant

Ellis

2020

Journal of Fish Biology

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The stomach contents of 640 starry smooth‐hound Mustelus asterias from the north‐east Atlantic were examined. The diet was dominated by crustaceans (98.8% percentage of index of relative importance, %IRI), with the two main prey species being hermit crab Pagurus bernhardus (34% IRI) and flying crab Liocarcinus holsatus (15% IRI). Ontogenetic dietary preferences showed that smaller individuals [20–69 cm total length (LT) n = 283] had a significantly lower diversity of prey than larger individuals (70–124 cm LT, n = 348); however, 18 prey species were found exclusively in smaller individuals and eight prey taxa were found exclusively in larger individuals. Larger commercially important brachyurans such as edible crab Cancer pagurus and velvet swimming crab Necora puber were more prevalent in the diet of larger individuals. Specimens from the North Sea ecoregion had a lower diversity of prey types for a given sample size than fish from the Celtic Seas ecoregion. Whilst cumulative prey curves did not reach an asymptote, this was primarily due to the high taxonomic resolution utilized and 95% of the diet was described by just seven crustacean taxa. The trophic level (TL) was calculated as 4.34 when species‐level prey categories were used. This fine‐scale taxonomic resolution resulted in a TL estimate close to a whole level above that estimated using wider taxonomic groupings. This large bias has important methodological implications for TL studies based on categorized prey data, particularly those of predatory fish.

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“…Due to the restrictions in the quantification of consumed prey volume, many trophic studies only use presence/absence data (e.g., Deagle et al., ; Harms‐Tuohy et al., ; Pinol, Mir, Gomez‐Polo, & Agusti, ). This might, however, result in an overestimation of small taxa that are abundant in the sediment, but with low trophic relevance, as they could, in the case of C. crangon , be passively acquired when shrimp ingest sediment to crush food in their stomach (Ansell et al., ; Deagle et al., ; Tiews, ). Multiple stomachs were pooled prior to analysis and data were subjected to rigorous filtering to allow for a semiquantitative estimation of proportions of prey DNA (Deagle et al., ; Lejzerowicz et al., ; Pompanon et al., ; Thomas, Deagle, Eveson, Harsch, & Trites, ).…”

Section: Discussionmentioning

confidence: 99%

DNA metabarcoding unveils multiscale trophic variation in a widespread coastal opportunist

et al. 2018

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A thorough understanding of ecological networks relies on comprehensive information on trophic relationships among species. Since unpicking the diet of many organisms is unattainable using traditional morphology‐based approaches, the application of high‐throughput sequencing methods represents a rapid and powerful way forward. Here, we assessed the application of DNA metabarcoding with nearly universal primers for the mitochondrial marker cytochrome c oxidase I in defining the trophic ecology of adult brown shrimp, Crangon crangon, in six European estuaries. The exact trophic role of this abundant and widespread coastal benthic species is somewhat controversial, while information on geographical variation remains scant. Results revealed a highly opportunistic behaviour. Shrimp stomach contents contained hundreds of taxa (>1,000 molecular operational taxonomic units), of which 291 were identified as distinct species, belonging to 35 phyla. Only twenty ascertained species had a mean relative abundance of more than 0.5%. Predominant species included other abundant coastal and estuarine taxa, including the shore crab Carcinus maenas and the amphipod Corophium volutator. Jacobs’ selectivity index estimates based on DNA extracted from both shrimp stomachs and sediment samples were used to assess the shrimp's trophic niche indicating a generalist diet, dominated by crustaceans, polychaetes and fish. Spatial variation in diet composition, at regional and local scales, confirmed the highly flexible nature of this trophic opportunist. Furthermore, the detection of a prevalent, possibly endoparasitic fungus (Purpureocillium lilacinum) in the shrimp's stomach demonstrates the wide range of questions that can be addressed using metabarcoding, towards a more robust reconstruction of ecological networks.

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Counting with DNA in metabarcoding studies: how should we convert sequence reads to dietary data?

Abstract: (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Cited by 83 publications

References 74 publications

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Diet composition of starry smooth‐hound Mustelus asterias and methodological considerations for assessing the trophic level of predatory fish

DNA metabarcoding unveils multiscale trophic variation in a widespread coastal opportunist

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